The invention relates to novel human membrane protein receptors for iysophospholipids and sphingolipids, and nucleic acids encoding these receptors. The invention is also directed to the use of these receptors in the discovery of agents that mediate or modulate apoptosis, cell proliferation, and other biological pathways in which phospholipid mediators are implicated.
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent phospholipid mediators with diverse biological activities. Their appearance and functional properties suggest possible roles in development, wound healing, and tissue regeneration. LPA and S1P appear to act in different cellular systems as paracrine, autocrine, and perhaps intracellular messengers. LPA and S1P are generated by complex enzymatic pathways from membranes of many different types of stimulated cells (Moolenar, W. H., J. Biol. Chem 270:12949 (1995); Spiegel and Milstein, J. Membrane Biol. 146:225 (1995); and Brindley et al., Biochem. Cell. Biol. 74:469 (1996)). LPA and S1P are both characterized by widespread cellular production, micromolar maximal concentrations in serum and some tissue fluids, high levels of binding to serun albumin and biodegradation by multiple enzymatic mechanisms (Fourcade et al., Cell 80:919 (1995); and Wang et al., J. Biol. Chem. 272:22030 (1997)). In extracellular fluids, these lipids are potent stimuli of cellular proliferation, differentiation, survival, adhesion, aggregation and other specific functions (Moolenaar et al., Curr. Opin. Cell Biol. 9:168 (1997; Gomez-Munoz et al., J. Biol. Chem. 210:26318 (1995); and Wu et al., J. Biol. Chem 270:11484 (1995)). LPA and S1P stimulate cellular proliferation directly by eliciting the serum response factor (SRF) and ternary complex factor (TCF) transcription factors, which together bind to and activate the serum response element (SRE) in promoters of many immediate-early genes (Hill, C. S. and Treisman, R., EMBO J. 14:5037-5042 (1995)).
The capacities of LPA and S1P to enhance cellular survival recently have been attributed in part to suppression of apoptosis (Cuvillier et al., J. Biol .Chem. 273:2910 (1998); Geotzl et al., J. Immunol. 162:2049 (1999); and Levine et al., Am. J. Physiol. 273:F575 (1997)). However, the complex mechanisms by which these lipids suppress apoptosis have not been elucidated fully. The specificity of LPA and S1P binding and initiation of signal transduction in numerous mammalian cells suggested that the actions of LPA and S1P are mediated by specific cell surface receptors (Van der Bend et al., EMBO J. 11:2495-2501 (1992)). The existence of G protein-coupled receptors (GPCRs) for LPA and S1P was suggested initially by specific ligand structural-dependence of their effects, ligand-induced desensitization of some cellular responses, and pertussis toxin inhibition of their cellular Ca++ mobilizing and proliferative activities (Durieux and Lynch, Trends Pharmac. Sci 14:249 (1993); and Goodemote et al., J. Biol. chem. 270:10272 (1995). Various GPCRs for LPA and S1P, have been identified (Hecht et al., J. Cell Biol. 135:1071 (1996); An et al., Biochem. Biophys. Res. Commun. 231:619-622 (1997); and Guo et al., Proc. Natl. Acad. Sci. USA 93:1436-1432 (1996).
The identification of additional LPA and S1P receptors is of interest because new receptors could provide additional tools for defmiing the mechanisms of LPA and S1P signaling and their physiological functions, and for identifying bioactive agents that simulate, modulate or mediate the functions of LPA and S1P.
In one of its several aspects, the invention provides isolated native sequences of the human Edg4 and Edg5 proteins, comprising the amino acid sequence of FIG. 1 (SEQ ID NO: 1) and FIG. 3 (SEQ ID NO:3), respectively.
In another aspect, the invention concerns an isolated Edg4 or Edg5 polypeptide, comprising an amino acid sequence having at least 85% sequence identity, preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity to the sequence of FIG. 1 (SEQ ID NO:1), or FIG. 3 (SEQ ID NO:3).
The invention also provides nucleic acids that encode the above-mentioned Edg4 and Edg5 polypeptides, as well as expression vectors and host cells comprising the Edg4- and Edg5-encoding nucleic acids. In one aspect, the isolated nucleic acid comprises DNA having at least 85% sequence identity, preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to a DNA molecule selected from the group consisting of (a) a DNA molecule encoding an Edg4 polypeptide having the sequence of FIG. 1 (SEQ ID NO: 1), (b) the complement of the DNA molecule of (a), (c) a DNA molecule encoding an Edg5 polypeptide having the sequence of FIG. 3 (SEQ ID NO:3), and (d) the complement of the DNA molecule of (c).
In another aspect, the isolated nucleic acid molecule encodes an Edg4 or Edg5 polypeptide and comprises DNA that hybridizes, preferably under stringent hybridization and wash conditions, to the complement of nucleic acid residues 85 through 1230 of FIG. 2 (SEQ ID NO:2), or to the complement of nucleic acid residues 1 through 1059 of FIG. 4 (SEQ ID NO:4).
In a still further aspect, the invention concerns an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity to either the amino acid sequence of FIG. 1 (SEQ ID NO:1) or FIG. 3 (SEQ ID NO:3), or the complement of such DNA.
The invention further provides screening assays for detecting the ability of a bioactive agent to simulate or modulate the activity of a lysophospholipid or sphingolipid for which the Edg4 or Edg5 polypeptide is a receptor. In the screening assay, a host cell comprising recombinant nucleic acid encoding an Edg4 or Edg5 polypeptide, and therefore expressing an Edg4 or Edg5 receptor, is contacted with a candidate bioactive agent, and the effects of the candidate bioactive agent directly on the cells and on lysophospholipid or sphingolipid cellular activities is determiined.
The invention additionally provides screening assays for detecting the ability of a bioactive agent to modulate the expression or activity of an Edg4 or Edg5 protein in a host cell, wherein a host cell comprising recombinant nucleic acid encoding an Edg4 or Edg5 polypeptide is contacted with a candidate bioactive agent, and the effects of the candidate bioactive agent on Edg4 or Edg5 expression or activity is determined.
In yet another embodiment, the invention concerns agonists and antagonists of a native Edg4 and/or Edg5 polypeptide. In a particular embodiment, the agonist or antagonist is an anti-Edg4 or anti-Edg5 antibody. In a further embodiment, the invention concerns a method of identifying agonists or antagonists of a native Edg4 and/or Edg5 polypeptide, by contacting the native Edg polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide, such as the binding of the polypeptide with its correlative ligand. In a still further embodiment, the invention concerns a composition comprising an Edg 4 and/or Edg5 polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier.